Various scientific articles patent or other publications are referenced throughout the specification. These publications are incorporated by reference herein to describe the state of the art to which this invention pertains.
Long a threat to human health, viral diseases have become a major public health concern in recent decades. Viral pandemics, for example polio and influenza, have been known historically, but the HIV pandemic that arose in the 1980s brought public health concerns about viruses to the forefront of modern research. Besides HIV, other viral diseases are emerging as serious public health concerns, including diseases caused by influenza, hepatitis, herpes viruses and papilloma virus.
Many of the viral diseases appear to be spreading: an example is herpes simplex virus Type 2 (HSV-2), the cause of genital herpes. Recent estimates are that more than 1 in 5 Americans age 12 and older tests seropositive for the HSV Type 2, with approximately 1 million new infections each year.
Statistics like those cited above have helped to spawn massive research efforts into antiviral treatments. Effective antiviral therapeutic agents are sought to combat the viruses mentioned above and many others. The dilemma faced by the researchers is based in the basic biological facts. Viruses, obligate intracellular parasites, while possessing some of their own unique molecular machinery, also rely heavily on cellular metabolism for their propagation. This poses a difficult challenge in that many potential metabolic targets to kill the virus are also toxic or lethal to the host cells, rendering the potential target useless for antiviral therapy.
A safe and effective antiviral agent must interrupt the life-cycle of the virus in some manner, such that the virus cannot replicate adequately, and the compound must have negligible or manageable toxicity to the host cells (or patient). In other words, the antiviral agent must be far more toxic to the virus than to the host cell. Another factor which has come to light relates to the long-term side-effects of certain antivirals, as well as the ability of the viruses to adapt or mutate to negate the effect of certain antiviral agents. Alternatively, more general compounds which boost or modulate immune system responses, such as interferons, may be used in combating viral disease, as may vaccines targeted to specific viruses.
In addition to searching for specific targets for antiviral agents, much research has focused on sources of new lead agents. While rational drug design has been used, in recent years the growth of fields like ethnobotany has been the direct result of the need for new sources of lead drugs. Ethnobotanists, and pharmacologists actively search the plant kingdom for potential active agents, particularly antimicrobial agents, including antiviral compounds.
Antiviral substances from plants are known (Vlietnick et al., 1997, Vanden Berghe et al., 1986). The primary focus of previous work in the field has not been on edible species, but instead on plants known for and used for medicinal purposes in various cultures including Indian (Banerjee, 1975, Elanchezhiyan et al., 1993), African (Beuscher et al., 1994, Ferrea et al, 1993), Bulgarian (Dimitrova et al., 1993, Serkedjieva et al., 1992), Paraguayan (Hayashi et al., 1990), Asian, including Chinese Indonesian and Japanese (Debiaggi et al., 1988, Kurokowa et al., 1993, Zheng, 1988). Methods of obtaining potentially useful plant materials typically has involved grinding or otherwise macerating a selected tissue, then preparing a crude extract from fresh or dried tissue or consuming the tissue directly.
These same studies have examined tissues from many different parts of the plant and stages of development, ranging from whole plant extracts to extracts from the aerial parts to those more specifically from leaves, stems, twigs, bark, flowers, buds, fruits, peels, seeds, roots, rhizomes, tubers, or bulbs. Thus far no studies have focused solely on substances that exist in the outermost layers of the plant, i.e., the epidermis and cuticle, or on substances deposited onto the surfaces of plants in an “epicuticular” layer.
The cuticle serves as an outer protective layer located external to the epidermis of most if not all plant parts, and consists of several different layers of non-cellular membranes. Peels or rinds as separated from fruits may consist of thickened membranes attached to a layer of epidermal cells. Bark is the most complex form of plant surface covering.
The cuticle is composed primarily of cutin, suberin, waxes and tannins along with a variety of other compounds. Cuticle composition varies from one variety of plant to another. The biosynthetic pathways leading to the complex compounds present are highly involved with many biosynthetic intermediates and alternate pathways present. Each component, such as the tannins or the waxes, may consist of a multitude of compounds, for example, apple wax contains well over 50 compounds including hydrocarbons, saturated and unsaturated fatty acids, primary and secondary alcohols, diols and hydroxy acids (Martin and Juniper, 1970; Cutler et al., 1982).
As to the spectrum of activity for plant-derived antivirals, many different viruses have been challenged with extracts of whole plants or various plant parts. Examples of viruses tested include HIV, polio virus, hepatitis, herpes viruses, such as HSV-1, HSV-2, cytomegalovirus and measles virus, influenza, rhinovirus, parainfluenza, vesicular stomatitis virus (VSV), vaccinia virus (VV), encephalitis, and African Swine Fever virus. Of the most frequently tested viruses, HSV-1 and HSV-2 have been challenged with numerous plant extracts. Some of the extracts have shown activity against these viruses, while others have not.
The field of plant-derived antiviral preparations is represented in the patent literature. U.S. Pat. No. 5,750,709 teaches a method for isolating therapeutic compositions from natural source materials in which the material is first de-waxed, de-fatted or de-oiled via supercritical fluid extraction. The de-fatted material is then further extracted to obtain the natural therapeutic composition. U.S. Pat. No. 4,871,540 teaches a process of producing an immune-modulating substance from the tissue of Zea mays. The substance is identified as a glycolipid and is expected to be useful in treating various disorders including asthma, rhinitis, hepatitis and AIDS and other disorders where an immuno-modulating substance would be effective. U.S. Pat. No. 5,411,733 claims methods for treating various viral diseases with a variety of plant extracts based on traditional medicinal plants. According to the teachings provided in this patent, the extracts were obtained via water or methanol extraction, as opposed to non-polar solvents. The compounds isolated were further subjected to and stable to boiling for 10 minutes. Presumably, they used the knowledge of the plants' traditional medicinal uses to identify which portions of the plant to use for preparation of the extracts. Most recently, U.S. Pat. No. 5,989,556 teaches compositions of plant extracts derived from traditional Chinese herbal medicines and useful in treating animals infected with hepatitis B, hepatitis C or HIV. The compositions taught therein are primarily mixtures of powdered herbs or water-based extracts or suspensions of these herbs and herb mixtures. They also teach a water-based extract which is subjected to and stable to boiling. They hypothesized that the active component was soluble in neutral aqueous solution; it was not soluble in alcohols, or common organic solvents, including hexane. The inventors suggest that the active component may be a simple organic acid.
Despite the amount of research and prior art, given the exemplary statistics cited above on the prevalence and rate of spread of HSV, antivirals from any source whatever, with low toxicity are needed to develop novel, safe and effective treatments for this disease.